Quorum sensing is a cell-cell communication process by which bacteria regulate behaviors as a function of cell population density. Quorum sensing involves the production, secretion, and detection of signal molecules termed autoinducers. While most autoinducers are highly specific for the species of bacteria that produces them, one autoinducer, termed AI-2, is produced and detected by many species, and functions as an interspecies signaling molecule. AI-2 is known to regulate numerous functions in Gram-positive bacteria, but in these cases, the AI-2 signaling cascade has not been investigated. The research in this proposal aims to define the role of AI-2 and the AI-2 signal transduction cascade in the Gram-positive pathogen Streptococcus pyogenes. To achieve these goals, I will develop genetic tools and use them to identify and characterize the S. pyogenes AI-2 quorum sensing signal transduction circuit, identify quorum sensing target genes, and determine the chemical moiety that functions as the S. pyogenes AI-2 signal. It has been a long standing goal in the quorum sensing field to control bacterial virulence by manipulating cell-cell communication. Results from this research will contribute to the development of such novel antimicrobial therapies. The specific aims are: 1. Identify the luxS/AI-2 quorum sensing signal transduction circuit. AI-2 controls production of SLS (hemolysin) and SpeB (cysteine protease) which are required for S. pyogenes virulence. Transposon mutageneses, followed by screens for altered production of hemolysin and protease will be used to identify genes required for AI-2 detection and signal transduction. 2. Identify genes regulated by AI-2. A library of random S. pyogenes promoters fused to a gfp gene, optimized for Gram-positive bacteria, will be constructed and introduced into a luxS S. pyogenes strain. Altered GFP production will be monitored by fluorescence activated cell sorting (FACS) following exogenous addition of synthetically prepared AI-2.;This screen should identify AI-2-activated and AI-2-repressed genes. 3. Determine the structure of the S. pyogenes AI-2 receptor and AI-2 molecule. A biochemical approach complementary to that in Aim 1 will employ radiolabeled AI-2 and cell fractionation to identify the S. pyogenes Al-2 receptor. The gene encoding the receptor will be cloned and the protein expressed and purified. X-ray crystallography will be used to determine the structure of all or a portion of th'e receptor with and without bound AI-2.